| Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus(PEDV),is an acute gastrointestinal disease.Before 2010,PED in China was sporadic and did not cause serious impact.At the end of 2010,PED broke out in China,causing 100%morbidity and 80-100%mortality in suckling piglets.At present,PED continues to be prevalent in China,and the vaccine immune protection is not effective,seriously affecting the development of China's pig industry.To study the genetic characteristics and pathogenicity of the prevalent PEDV,the whole genome sequencing and analysis of PEDV SH and PEDV YZ strains was conducted in this study,and the two strains were determined to be subgroup G2a epidemic strains.In addition,12-aa deletion in the N protein of PEDV SH strain was found for the first time.The epitope NEP-1C9 coincidentally locates in the deletion region.Infection experiments were carried out on newborn piglets with PEDV SH strain,and it was determined to be highly pathogenic to newborn piglets,causing 100%morbidity and mortality.To further study whether the epitope deletion affects the immunogenicity of PEDV SH strain,the mice were immunized with inactivated PEDV SH and PEDV YZ,respectively,and the induced antibody levels were detected using the polypeptides and recombinant N protein.The results demonstrated that NEP-1C9 had good immunogenicity,PEDV SH and PEDV YZ could be distinguished by the presence or absence of the corresponding antibody;The N protein immunogenicity of the PEDV SH strain was not significantly affected by the deletion of NEP-1C9.which provided a new idea for the development of PEDV vaccine.The details are as follows:1.Whole genome sequencing and analysis of PEDV strainsIn this study,the whole genome amplification and sequencing of strains PEDV SH and PEDV YZ isolated from Shanghai and Yangzhou were performed with segmentation overlapping method.Genetic evolution analysis showed that both of them belonged to subgroup G2a and were epidemic variant strains,which had a closer phylogenetic relationship with Chinese epidemic strains post-2010,compared with the classical strains.The whole genome identity analysis showed that the identity of PEDV SH,PEDV YZ and the mutant strains post-2010 was 98.3%,and the identity with the classical strain was only 96.5-97.2%.The identity of S protein between PEDV SH and PEDV YZ was 99.9%.The identity analysis revealed that both of them had 96.5%,95-98.5%,92.0-93.1%identity with Chinese isolates post-2010,and Japanese,Korean,American isolates,classical strains,respectively.Compared with CV777,both of them had 15 bases-deletion and 6 bases-insertion,in addition to many point mutations,resulting in multiple changes of neutralization epitope and potential N-glycosylation sites in the S protein.Besides,36 consecutive bases deletion in the N gene of PEDV SH strain was found for the first time,compared with other strains.2.Pathogenicity test of PEDV SH strain on newborn pigletsIn this study,3-day-old piglets were orally infected with the PEDV SH strain and the pathogenicity of PEDV SH was investigated through clinical symptoms and pathological observation,immunohistochemistry and viral load determination.The piglets developed mild diarrhea at 18 hpi.The typical symptoms,vomiting and watery diarrhea were obvious at 36 hpi.During 69-84 h,the piglets gradually died of dehydration.Pathological examination revealed thinning of the intestinal wall,dilatation of the intestinal cavity,yellow-white liquid and milk in the intestine,mesenteric lymph node congestion,and the stomach was filled with undigested white milk-like substance.Pathological microscopic observation showed that jejunum intestinal villi seriously atrophied,shed,and accompanied by inflammatory cell infiltration.Brown-stained PEDV-positive particles were detected in the cytoplasm of jejunal villus epithelial cells by immunohistochemistry.Large amounts of the virus could be detected in feces at 24 hpi.No symptoms,lesions or virus particles were observed or detected in the control group.This study indicated that the PEDV SH strain was highly pathogenic to newborn piglets and could cause 100%morbidity and mortality.3.Immunogenicity analysis of PEDV SH strainThere is a 12 aa-deletion(aa 399-410)in the N protein of PEDV SH strain,in which the epitope NEP-1C9(aa 398-406)coincidentally locates.To study whether the epitope deletion affects the immunogenicity of PEDV SH strain,we artificially synthesized four deleted polypeptides based on the epitope sequences of different strains,and identified the polypeptides reactogenicity by indirect ELISA to determine whether they could be used as coating antigens.The mice were immunized with inactivated PEDV SH and PEDV YZ,respectively,and after four immunizations,serum was collected,and the antibody levels were detected using the above polypeptide and recombinant N protein.The results showed that the polypeptides had good reactogenicity and could be used as coating antigens.The I-ELISA results demonstrated that the PEDV YZ strain could induce antibodies against the N protein and deleted polypeptide in mice.while the NEP-1C9 deletion strain,PEDV SH,could induce the production of N protein antibodies,but could not produce corresponding antibodies against the deleted polypeptide.This study indicated that the N protein immunogenicity of the PEDV SH strain was not affected by the deletion of NEP-1C9. |
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